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Team:University College London/LabBook/Week5
From 2012.igem.org
Contents |
Monday (9.7.12)
Aim: Transformation of Key BioBricks
Method
(LOGO) Transformation Protocol 2
Step 1 – Thawing Cells: Use W3100 cell line created in Week 2 (Expt 2.1) Step 3 – Addition of BioBrick: To each 2ml eppendorf, add 1ul of the following BioBricks. Include an extra tube as a control, with no BioBrick added.
| Function | Module | ||
|---|---|---|---|
| BioBrick | BBa_K123003 | Oestrogen Receptor | Detection |
| BBa_K123002 | Oestrogen Response Element | Detection | |
| BBa_K398108 | Salt Tolerance Cluster | Salt Tolerance | |
| BBa_J23100 | Constitutive Promoter | ||
| BBa_J23107 | Constitutive Promoter | ||
| BBa_R0040 | TetR Repressible Promoter | Buoyancy | |
| BBa_J23106 | Constitutive Promoter | ||
| BBa_B0034 | Ribosome Binding Site (RBS) | All | |
| Control | (No BioBricks) | ||
Step 9 – Plating samples on Agar Plates: The table below indicates the chosen inoculation volume (two for each BioBrick) and the correct gel antibiotic concentration for all samples.
| Samples | Volume Inoculated | Antibiotic in Gel (ug/ml) | |
|---|---|---|---|
| BioBrick | BBa_ K123003 | 20ul | Ampicillin(50ug/ml) |
| 200ul | |||
| BBa_ K123002 | 20ul | ||
| 200ul | |||
| BBa_B0034 | 20ul | ||
| 200ul | |||
| BBa_J23100 | 20ul | ||
| 200ul | |||
| BBa_J23107 | 20ul | ||
| 200ul | |||
| BBa_R0040 | 20ul | ||
| 200ul | |||
| BBa_J23106 | 20ul | ||
| 200ul | |||
| BBa_ K398108 | 20ul | Chloramphenicol (25ug/ml) | |
| 200ul | |||
| Control | Positive (No BioBrick) | 36ul | No Antibiotic |
| Negative (No BioBrick) | 36ul | 2x Ampicillin(50ug/ml)
1x Chloramphenicol (25ug/ml) | |
Tuesday (10.7.12)
Aim - Check results from Transformation
Results: The table below indicates whether or not there was growth on each plate. Included is an image demonstrating the growth noted for BBa_K123003, BBa_K398108, and the Positive Control
| Samples | Volume Inoculated | Growth/No Growth | |
|---|---|---|---|
| BioBrick | BBa_ K123003 | 20ul | Growth |
| 200ul | Growth | ||
| BBa_ K123002 | 20ul | No Growth | |
| 200ul | No Growth | ||
| BBa_B0034 | 20ul | No Growth | |
| 200ul | No Growth | ||
| BBa_J23100 | 20ul | No Growth | |
| 200ul | No Growth | ||
| BBa_J23107 | 10ul | No Growth | |
| 2000ul | No Growth | ||
| BBa_R0040 | 20ul | No Growth | |
| 200ul | No Growth | ||
| BBa_J23106 | 20ul | No Growth | |
| 200ul | No Growth | ||
| BBa_ K398108 | 20ul | No Growth | |
| 200ul | Growth | ||
| Control | Positive (No BioBrick) | 36ul | Growth |
| Negative (No BioBrick) | 36ul | No Growth | |
Conclusion: Only BBa_K398108 and BBa_K123003 had successfully transformed. This is a serious setback, which suggests our cell competency is not as high as we first anticipated. Method
(LOGO) Picking Colonies
Step 2 - Inoculating Colonies into a Selective Broth: The table below indicates the volume of broth and the concentration of antibiotic used for the BioBrick.
| Samples | Volume Inoculated | Broth (ml) | Antibiotic (ug/ml) | |
|---|---|---|---|---|
| BioBrick | BBa_ K123003 | 20ul | Lysogeny Broth (5) | Ampicillin(50ug/ml) |
| 200ul | ||||
Wednesday (11.7.12)
Aim 1 – Check Results of Picking Colonies of BBa_K398108.
Result: The broth was cloudly, indicating there has been sufficient growth of bacteria to proceed to purification (miniprep) and an Analytical Restriction Digest to determine the presence of the correct BioBrick.
Method (LOGO) Miniprep Protocol 1 – Qiagen (LOGO) Restriction Digest
Step 2 – Setting up Digests and Controls: The protocol describes the recipe for (i) Digested Plasmid and (ii) Uncut Control. The table below indicates that an uncut and an Spe1/Xba1 digested sample be set up for each BioBrick.
| Samples | Recipe | Enzyme Used | Buffer | |
|---|---|---|---|---|
| BioBrick | BBa_K398108 | Digested Plasmid | Spe1 and Xba1 | 4 |
| Undigested Plasmid | None | |||
(LOGO) Gel Electrophoresis
Results: In Lane 1 and 2 we expect a product equivalent to the size of the PSB1C3 plasmid backbone and BBa_K398108 insert (2914bp) long, indicated by the position of A. The absence of this band, and the presence of other bands indicates that this transformation was unsuccessful. In Lane 3 and 4 we would expect a product for the BBa_K398108 insert (844bp) as indicated by B, and a product for the PSB1C3 plasmid backbone (2070bp) as indicated by C. Both products are absent, further supporting the failure of the transformation. In Lane 5 and 6 we would expect to see a similar product to Lanes 1 and 2, except for any secondary effect that the conformation of the uncut plasmid may have on its migration. Such a product was not found.
(Insert Gel PIC)
Conclusion: This transformation failed, or contamination has occurred since the colony picking.
(LOGO) Nanodrop
| BioBrick | 260 | 280 |
|---|---|---|
| BBa_K398108 | 173.8 | 186.6 |
Aim 2 - Picking Colonies for BBa_K123003. With the exception of BBa_K398108, which has already been analysed, BBa_K123003 was the only other BioBrick to show growth in the original Expt 5.1 cohort. Colonies from the Agar Plate will be selected and cultured, with the intent of purifying the plasmid for detecting (Restriction Enzyme Digest and Gel Electrophoresis) the presence of the correct BioBrick.
Method (LOGO) Picking Colonies
Step 2 - Inoculating Colonies into a Selective Broth: The table below indicates the volume of broth and the concentration of antibiotic used for inoculating the BioBrick.
4.1
5.3
5.2
